SIRT6 attenuates LPS‐induced inflammation and apoptosis of lung epithelial cells in acute lung injury through ACE2/STAT3/PIM1 signaling

Abstract Background Acute lung injury (ALI) is a severe and fatal respiratory disease. SIRT6 exerts pivotal activities in the process of lung diseases, but whether SIRT6 impacts ALI has not been covered. Methods Lentivirus recombinant expressing vector SIRT6 gene (Lent‐SIRT6) was constructed in mice, and there were control, lipopolysaccharide (LPS), LPS + Vehicle, and LPS + Lent SIRT6 groups. RT‐qPCR and western blot detected SIRT6 expression in lung tissues. HE staining observed pathological alternations in lung tissues. Wet‐to‐dry ratio of the lungs was then measured. The cell count of bronchoalveolar lavage fluid (BALF) was evaluated. Serum inflammation was examined with enzyme‐linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and western blot were to measure apoptosis. Western blot tested the expression of ACE2/STAT3/PIM1 signaling‐associated factors. At the cellular level, LPS was used to induce lung epithelial cells BEAS‐2B to establish cell injury models. SIRT6 was overexpressed and ACE2 expression was inhibited by cell transfection, and the mechanism of SIRT6 in LPS‐induced lung injury model was further explored by Cell Counting Kit‐8 (CCK‐8), western blot, quantitative reverse‐transcription polymerase chain reaction, TUNEL, and other techniques. Results The results of animal experiments showed that SIRT6 overexpression could reduce LPS‐induced lung pathological injury, pulmonary edema, and BALF cell ratio and attenuate LPS‐induced inflammatory response and cell apoptosis. In the above process, ACE2, STAT3, p‐STAT3, and PIM1 expression were affected. In cell experiments, SIRT6 expression was reduced in LPS‐induced BEAS‐2B cells. Inhibition of ACE2 expression could reverse the inhibitory effect of SIRT6 overexpression on ACE2/STAT3/PIM1 pathway, and cellular inflammatory response and apoptosis. Conclusion SIRT6 eased LPS‐evoked inflammation and apoptosis of lung epithelial cells in ALI through ACE2/STAT3/PIM1 signaling.


| INTRODUCTION
Acute lung injury (ALI) is a clinical syndrome of acute respiratory failure caused by various intrapulmonary and extrapulmonary pathogenic factors. 1 ALI is a severe respiratory disease featured by uncontrolled oxidative stress, pulmonary edema, inflammation, neutrophil infiltration, and so forth. 2 Currently, the fatality rate of ALI remains 40%-60%. 3 Therefore, it is very important to actively seek effective therapy for ALI.
Seven sirtuin subtypes (SIRT1-SIRT7) widely expressed in mammalian genomes play important roles in various physiological processes such as cell growth and apoptosis. 4 Recent studies have confirmed that SIRT6 plays a key regulatory role in gene transcription, metabolism, maintenance of genomic stability, and telomere integrity. 5,6 A previous study has shown that SIRT6 expression is decreased in sepsis-induced lung injury. Overexpression of SIRT6 can alleviate lipopolysaccharide (LPS)-induced injury of vascular endothelial cells. 7 A study has shown that SIRT6 has a protective effect on radiation-induced lung injury. 8 In addition, a number of studies have reported that SIRT1 has a protective effect on various types of lung injury, LPSinduced lung injury is also included. [9][10][11][12] However, whether SIRT6 can play a role in LPS-induced ALI is unknown.
A recent study has shown that SIRT6 has a protective effect on cholesterol crystal-induced endothelial dysfunction by regulating ACE2 expression. 13 SIRT6 can regulate ACE2 expression in rat aortic membrane fibroblasts. 14 These results have implied that ACE2 expression can be modulated by SIRT6. ACE2 is the binding receptor of the severe acute respiratory syndrome coronavirus 2, so activation of ACE2 has an important protective effect on lung injury. 15 Studies have shown that ACE2 can relieve lung injury caused by pulmonary hypertension by inhibiting downstream STAT3 signaling pathway to exert antioxidant activity. 16,17 Moreover, studies have clearly demonstrated that inhibition of STAT3 expression can significantly reduce LPS-induced ALI. 18 20,21 Therefore, in this article, we mainly discuss the role of SIRT6 in ALI and the mechanism of SIRT6 in lung injury through the regulation of ACE2/STAT3/PIM1 signal.

| Western blot
Bicinchoninic acid (BCA) kit (Beyotime) was to confirm protein concentration following isolation of proteins from cells utilizing RIPA buffer (Beyotime) in light of the manufacturer's instructions. Then polyvinylidene fluoride membranes were to transfer 30 μg of protein per well loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Primary antibodies and horseradish peroxidaseconjugated secondary antibody (Protein Tech Group) at a dilution of 1:5000 were supplemented to the membranes impeded with 5% of skim milk powder respectively at 4°C for overnight and for 1 h at room temperature. Animalrelated proteins were developed by using the film developer. For cellular-related proteins, ECL substrates (Thermo Pierce) were used to protein development. And ImageJ (Version146) was to develop and analyze the density of immunoblotting bands.

| Histopathological examination (HE) staining
Following immobilization with 10% formalin solution for 24 h at 4°C, hematoxylin and eosin (H&E) were to stain paraffin-embedded tissues which were cut into 5 μmthick sections. The lung injury was assessed by histological examination. According to the histological injury parameters, including lung tissue edema, alveolar septal cell exudation, congestion, and alveolar hemorrhage, lung tissue injury was divided into four grades: normal (0 points), mild (1 points), moderate (2 points), and severe (3 points), and semi-quantitative analysis of the injury was performed. The total lung injury score was calculated as the summation of individual scores of each item.
2.5 | Measurement of wet-to-dry ratio (W/D) of the lungs Following dissection in mice, "wet" weight of the right lungs was acquired. Then "dry" weight was acquired after the lungs were dried at 60°C for 48 h. The extent of pulmonary edema was evaluated using the Wet/Dry ratio.

| Giemsa staining
The 0.3 mL precooled saline was to irrigate the lungs of mice for three times and the harvested BALF was centrifugated at 2000 r/min for 5 min. Bronchoalveolar lavage fluid (BALF) was applied to the slide, which then dried naturally. About 2-3 drops of Giemsa staining solution covered the entire specimen smear for 1-2 min. An equal amount of phosphate buffer (pH 6.4) was added to the slide. The slide was gently shook and thoroughly mixed with Giemsa staining solution for 3-5 min.
Finally, the slides were washed, dried, stained, and examined for photo analysis. Then cell counter was to examine total cells, neutrophils, and macrophages proportion in BALF.
another three mice of each group using ELISA kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocols.

| Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining
The lung cells were prepared, and cell apoptosis was measured by the Apoptosis Fluorescein Detection Kit (Millipore). The cells were fixed in 4% paraformaldehyde for 5 min. Terminal deoxyribonucleotidyl transferase and deoxyuridine triphosphate was to cultivate 4% paraformaldehyde-immobilized cells. Nuclear staining was performed with DAPI (Invitrogen). Image-Pro Plus 6.0 software was to analyze the results captured by a fluorescence microscope.

| Cell Counting Kit-8 (CCK8) assay
After indicated treatment of a total of 5000 cells seeded into 96-well plates accordingly, the medium was discarded, and each well of the plate was supplemented with 100 µL fresh medium with 10% CCK8 solution (Dojindo) for 2 h. A microplate reader (Thermo Fisher) was to measure OD450 nm value.

| Statistical analysis
Data analyzed by SPSS 22.0 software were denoted as mean ± standard deviation (SD). Unpaired Student's ttest or one-way analysis of variance (ANOVA) analysis followed by Tukey post hoc test was to compare group differences. It was regarded to possess statistical significance when p value < .05. The experiment was repeated at least three times.

| Overexpression of SIRT6 alleviated lung pathological injury in LPS-induced mice
First, SIRT6 expression in lung tissues was examined with RT-qPCR and Western blot. The results manifested that SIRT6 expression was prominently declined in the LPS group relative to the control group. By contrast with LPS + Vehicle group, SIRT6 expression was distinctly fortified in the LPS + Lent-SIRT6 group ( Figure 1A,B). Subsequently, the results from HE staining implied that the pathological state of lung tissue in the LPS group was more serious than that in the control group. SIRT6 expression did not change between LPS and LPS + Vehicle groups. Relative to the LPS and LPS + Vehicle groups, the pathological status of lung tissues was reduced in the LPS + Lent-SIRT6 group ( Figure 1C,D).

| Overexpression of SIRT6 reduced pulmonary edema and the proportion of cells in BALF in LPS-induced mice
Through assessment of lung W/D ratio, it was noted that in the LPS group, the lung edema was increased by contrast with the control group. Relative to the LPS and LPS + Vehicle groups, pulmonary edema was reduced in F I G U R E 5 Overexpression of SIRT6 regulated ACE2/STAT3/PIM1 signaling in LPS-induced mice. Western blot detected the expression of ACE2/STAT3/PIM1 signaling-related proteins in lung tissues. **p < .01, ***p < .001 vs. Control; & p < .05, && p < .05, &&& p < .001 vs. LPS; # p < .05, # p < .01 vs. LPS + Vehicle. LPS, lipopolysaccharide. the LPS + Lent-SIRT6 group (Figure 2A). Through Giemsa staining, we also found that total cell, neutrophil, and macrophage proportion were increased in the LPS group by contrast with the control group. Total cell, neutrophil, and macrophage proportion were decreased in the LPS + Lent-SIRT6 group by contrast with LPS and LPS + Vehicle groups ( Figure 2B).

| Overexpression of SIRT6 attenuated inflammatory response and lung cell apoptosis in LPS-induced mice
ELISA results elucidated that TNF-α, IL-1β, and IL-6 in the LPS group were notably aggrandized by contrast with the control group. Relative to LPS and LPS + Vehicle groups, TNF-α, IL-1β, and IL-6 expressions were decreased in the LPS + Lent-SIRT6 group (Figure 3). TUNEL and western blot results illuminated that apoptosis level was elevated in the LPS group, which was also evidenced by increased Bax, Cleaved caspase3 expression, and lessened Bcl2 expression by contrast with the control group. Relative to LPS and LPS + Vehicle groups, the apoptotic cells in the LPS + Lent-SIRT6 group were decreased, which was evidenced by declined Bax, cleaved caspase3 expression, and augmented Bcl2 expression ( Figure 4A,B).

| Overexpression of SIRT6 regulated ACE2/STAT3/PIM1 signaling in LPSinduced mice
In the experiment, we found abnormal expression of ACE2/STAT3/PIM1 signal-associated proteins in lung tissues after overexpression of SIRT6. Western blot results elaborated that p-STAT3 and PIM1 expression in the LPS group were increased, while ACE2 expression was cut down relative to the control group. p-STAT3 and PIM1 expression were decreased in LPS + Lent-SIRT6 group, while ACE2 expression was fortified obviously by contrast with LPS and LPS + Vehicle groups ( Figure 5).

| The regulation of SIRT6 on ACE2/ STAT3/PIM1 signaling in LPS-induced lung epithelial cells
Next, we will discuss the mechanism in cell experiments. Lung epithelial cells were induced by different concentrations of LPS and cell activity was detected by CCK8. The results showed that LPS treatment induced cell activity injury ( Figure 6A). Subsequently, western blot results revealed that SIRT6 expression was notably lessened upon exposure to 300 μg/mL of LPS ( Figure 6B). Therefore, 300 μg/mL LPS was adopted for the ensuing experiments. The interference plasmid of ACE2 was constructed, and the plasmid transfection efficacy was detected by RT-qPCR. The interference efficacy siRNA-ACE2-1 was better than others, as shown in Figure  into. Western blot results uncovered that p-STAT3 and PIM1 expression were augmented in the LPS group, while ACE2 expression was declined relative to the control group. p-STAT3 and PIM1 expressions were cut down, and ACE2 expression was aggrandized in LPS + Ov-SIRT6 group relative to LPS + Ov-NC group. p-STAT3 and PIM1 expression in LPS + OV-SIRT6 + siRNA-ACE2 group were increased but were lower than that in the LPS group, while ACE2 expression was decreased but was higher than that in the LPS group relative to LPS + Ov-SIRT6 + siRNA-NC group ( Figure 7B).
3.6 | SIRT6 attenuated inflammatory response and apoptosis of LPS-induced lung epithelial cells through ACE2/STAT3/ PIM1 signaling ELISA was to test contents of TNF-α, IL-1β, and IL-6 in the culture medium of lung epithelial cells. It turned out that TNF-α, IL-1β, and IL-6 expression in the LPS group were elevated by contrast with the control group. TNF-α, IL-1β, and IL-6 expression were markedly lessened in LPS + Ov-SIRT6 group by contrast with LPS + Ov-NC group. After further inhibition of ACE2 expression, in LPS + Ov-SIRT6 + siRNA-ACE2 group, TNF-α, IL-1β, and IL-6 expression were increased, whereas lower than in the LPS group (Figure 8). Similarly, by contrast with the control group, apoptosis level was boosted in the LPS group, evidenced by fortified Bax, Cleaved caspase3 expression, and declined Bcl2 expression. The apoptosis level of the LPS + Ov-SIRT6 group was attenuated, Bax and Cleaved caspase3 expressions were reduced, and Bcl2 expression was increased by contrast with the LPS + Ov-NC group. Relative to the LPS + Ov-SIRT6 + siRNA-NC group, the level of apoptotic cells in the LPS + Ov-SIRT6 + siRNA-ACE2 group was increased, whereas lower than that in the LPS group, and Bax, Cleaved caspase3 expression levels were increased but lower than those in the LPS group, and Bcl2 expression levels were decreased but higher than those in the LPS group ( Figure 9A,B).

| DISCUSSION
LPS is widely recognized as a lung injury inducer and has been applied in ALI model construction. 23,24 Direct injection of LPS into lung tissues may result in increased accumulation of peripheral inflammatory cells, increased pulmonary capillary permeability, alveolar and interstitial edema, and eventually lead to acute inflammatory response. 25 In this study, we induced mice and lung epithelial cells with LPS in vitro and in vivo to establish ALI models. We found that the pathological state of the lungs of mice induced by LPS was severe, with edema in the lungs and enhanced proportion of total cells, neutrophils, and macrophages, as well as inflammation and apoptosis in the lung tissues and lung epithelial cells, suggesting the successful construction of lung injury model. LPS plays a key role in the apoptosis of pulmonary endothelial and epithelial cells, 26,27 and pulmonary epithelial cell apoptosis is one of the key factors in the formation of blood-brain barrier in pulmonary fibrosis. 28 Therefore, it is of great significance F I G U R E 8 SIRT6 attenuated inflammatory response of LPS-induced lung epithelial cells through ACE2/STAT3/PIM1 signaling. ELISA examined TNF-α, IL-1β, and IL-6 activities in the culture medium of lung epithelial cells. ***p < .001 vs. control; ### p < .001 vs. LPS + Ov-NC; &&& p < .001 vs. LPS + Ov-SIRT6 + siRNA-NC. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; siRNA, small interfering RNA.
to effectively inhibit the inflammation and apoptosis of lung epithelial cells in ALI.
It was discovered that SIRT6 expression was remarkably reduced in LPS-induced animals and cells. SIRT6 is a histidine deacetylase that plays a role in injury-related diseases induced by inflammation. 29,30 A previous study has showed that SIRT6 has a synergistic effect on SIRT1. 31 And a number of studies have reported that SIRT1 has a protective effect on various types of lung injury, LPS-induced lung injury is also included. 9,10 In our experiment, it was found that overexpression of SIRT6 could significantly improve the pathological state and pulmonary edema of LPS-induced mice, and inhibit pulmonary inflammation and pulmonary cell apoptosis, which implied that overexpression of SIRT6 could improve the symptoms of ALI disease. Studies have shown that SIRT6 eases sepsis-elicited acute respiratory distress syndrome via promoting macrophage M2 polarization. 32 Moreover, in sepsis-evoked ALI, SIRT6 positively regulates Nrf2 expression, activates Nrf2-mediated anti-inflammatory and antioxidant enzymes, and can effectively alleviate LPS-induced inflammation of HU-VECs. 7 These reports were in agreement with our experimental results. The study has shown that ACE2 has a protective effect on LPS-induced ALI in mice by inhibiting LPS-TLR4 pathway. 15 Severe acute respiratory syndrome coronavirus receptor ACE2 protects lungs from damage. 33 It has been shown that SIRT6-ACE2 has a regulatory effect, and SIRT6 can regulate the expression of ACE2 in rat aortic membrane fibroblasts. 14 In our experiment, it was found that when SIRT6 was upregulated, the expression of ACE2 in mouse lung tissues and lung epithelial cells was significantly increased. Therefore, we hypothesized that SIRT6 played a role in ALI by regulating ACE2. Moreover, ACE2 is upregulated by IL-6 through STAT3 signaling in synovial tissues. 34 KEGG Pathway showed that STAT3 activation could activate the expression of downstream target gene PIM1. In addition, PIM1 targeting can reduce apoptosis and oxidative stress in CCL4-induced ALI in mice. 35 In this experiment, we found that the expression of p-STAT3 and PIM1 in cells was significantly inhibited after SIRT6 expression was knocked down. At this point, the inflammatory response and apoptosis were suppressed. Inhibition of ACE2 could reverse the inhibitory effect of SIRT6 overexpression on downstream STAT3 and PIM1, and reverse the protective effect of SIRT6 overexpression on cells. See Figure 10 for the specific mechanism diagram.
Our article also has some limitations. In the process of mechanism study, we only inhibited the expression of ACE2, but no experiments have been conducted to verify ACE2 overexpression. In the following experiments, we will further overexpress ACE2 so as to explore the regulatory mechanism of SIRT6.

| CONCLUSION
To sum up, we found that SIRT6 mitigated LPS-elicited inflammation and apoptosis of lung epithelial cells in ALI through ACE2/STAT3/PIM1 signaling. Our paper offered a solid theoretical basis for the therapy for sepsis-induced ALI.
AUTHOR CONTRIBUTIONS Juan Yang and Xing Chen performed the research. Xing Chen designed the research study. Juan Yang contributed essential reagents or tools. Xing Chen analysed the data. Juan Yang and Xing Chen wrote the paper. All authors have read and approved the final manuscript.

CONFLICTS OF INTEREST STATEMENT
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

ETHICS STATEMENT
All animal procedures were operated in light of the NIH Guide for the Care and Use of Laboratory Animals, approved by the ethical guidelines of Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University (approve number: NO.2022-016), and were conducted in light of the ARRIVE guidelines.

CONSENT FOR PUBLICATION
All the authors agreed to be published.